Ataxia-telangiectasia (AT) is a human autosomal recessive disease that exhibits progressive neuromuscular problems, immunodeficiencies, a high incidence of lymphoreticular cancer, and sensitivity to ionizing radiation [Taylor, "Cytogenetics of ataxia telangiectasia, IN: Bridges and Harnden (eds.) Ataxia-telangiectasia--a cellular and molecular link between cancer, neuropathology and immune deficiency, pp. 53-82 (Wiley, Chichester 1988); Boder, "Ataxia telangiectasia an overview," IN: Gatti and Swift (eds.), Ataxia telangiectasia: genetics, neuropathology and immunology of a degenerative disease of childhood, pp. 1-63 (Alan R. Liss; New York, 1985); and Morrell et al., J. Natl. Cancer Inst., 77: 89-92 (1986)]. AT heterozygotes, which constitute as much as 3% of the human population, have been reported to have an increased risk of cancer after exposure to ionizing radiation.
Cells from patients with AT display two hallmark characteristics: hypersensitivity to the killing effects of ionizing radiation [Taylor et al., Nature, 258: 427-429 (1975)] and resistance to the inhibiting effects of ionizing radiation on the rate of DNA synthesis, that is, radioresistant DNA synthesis [Young and Painter, Hum. Genet., 82: 113-117 (1989)]. Thus, the identification of genes that are responsible for those abnormalities would greatly further the understanding of human radiosensitivity and the regulation of DNA replication after radiation-induced DNA damage. The characteristic of AT cells to exhibit radioresistant DNA synthesis has been used to establish the presence of several complementation groups within this disease [Jaspers and Bootsma, PNAS (USA), 79: 2641-2644 (1982); Murnane and Painter, PNAS (USA), 79: 1960-1963 (1982); Jaspers et al., Cytogenet. Cell Genet., 49: 259-263 (1988)].
Despite extensive investigation, the underlying defects responsible for the pleiotropic abnormalities presented by AT remain unknown. Genetic linkage analysis [Gatti et al., Nature, 336: 577-580 (1988); McConville et al., Nucl. Acids Res., 18: 4335-4343 (1990a); McConville et al., Hum. Genet., 85: 215-220 (1990b); Sanal et al., Am. J. Hum. Genet., 47.: 860-866 (1990); and Ziv et al., Genomics, 9: 373-375 (1991)]0 and chromosome transfer studies [Lambert et al., PNAS (USA), 88: 5907-5911 (1991)] have shown that the gene(s) associated with three complementation groups are all located at the chromosomal region 11q22-q23.
Complementation groups A (AT-A) and C (AT-C) have been mapped by genetic linkage analysis [Gatti et al. 1988; McConville et al. 1990a and 1990b; Sanal et al. 1990; Ziv et al. 1991; and Foroud et al., Am. J. Hum. Genet., 49: 1263-1279 (1991)]. Using families from mixed complementation groups, two groups of investigators independently reported linkage of the AT gene(s) to two separate regions, one of which is near THY1 [McConville et al. 1990a and 1990b; Sanal et al. 1990; Foroud et al. 1991]. It was subsequently determined that the genes for AT-A and AT-C are located within the more centromeric of these two regions [McConville et al. 1990a; Sanal et al. 1990; Foroud et al. 1991], although it was concluded that the AT gene in a small subset of families could map to the second locus near THY1 [Sanal et al. 1990]. A recent study by Gatti et al. [in paper entitled " Ataxia-telangiectasia: linking evidence for genetic heterogeneity," presented at AACR (American Association for Cancer Research) Special Conference "Cellular Responses to Environmental DNA Damage" in Banff, Alberta (Canada) Dec. 1-6, 1991] which excluded families in complementation groups A and C, concluded that a gene for an additional complementation group does show linkage to the region near THY1. The gene for complementation group D (AT-D) was a likely candidate for linkage near THY1 as complementation group D is the next most common complementation group for AT after A and C [Jaspers et al. 1988].
Functional complementation has been used to prove the identity of several genes that provide resistance to various DNA-damaging agents [van Duin et al., Cell, 44: 913-923 (1986); Thompson et al., Mol. Cell Biol., 10: 6160-6171 (1990); and Weeda et al., Mol. Cell Biol., 10: 2570-2581 (1990)].
Using the sensitivity of AT cells to ionizing radiation, Kapp and Painter [Int. J. Radiat. Biol., 56: 667-675 (1989)] attempted to complement the defect in an AT cell line (AT5BIVA) from complementation group D (AT-D) by transfection with a human cosmid library containing a selectable neo gene. The combined selection by ionizing radiation and G418 resulted in the isolation of an AT cell line (1B3) that is partially resistant (approximately 50% of normal) to ionizing radiation and produces fewer radiation-induced chromosome aberrations, but retains the AT characteristic of radioresistant DNA synthesis. Southern blot analysis demonstrated that the 1B3 cell line contains at least three cosmids that appear to be integrated in tandem and coamplified [Kapp and Painter 1989, id.]. Transfer of cellular DNA containing those integrated cosmid sequences to AT5BIVA cells produced cell clones with radioresistance similar to that of 1B3 cells, indicating that a gene within the cosmids complements the defect in the AT-D group [Kapp and Painter 1989, id].
Functional complementation of that same cell line (AT5BIVA) has been accomplished by microcell-mediated chromosome transfer from mouse-human hybrids [Lambert et al. 1991]. That study showed that the gene for AT-D was within a recombinant chromosome that contained a human chromosome 11q23 fragment that was telomeric to the AT-A and AT-C linkage region. However, the report did not state whether that mouse-human hybrid also contained the chromosome 11q23 region telomeric to THY1.
The inventors hereof cloned DNA from a fragment of an AT gene for complementation group D. That DNA was of value as a probe to find and clone an entire AT gene for complementation group D, that is, an ATDC gene, and to identify a region in 11q23 where the ATDC gene is located. Prior to the cloning described herein, basic research and clinical work on AT had moved very slowly as there had been no specific gene recognized for AT. Further, there had been no simple biochemical or laboratory test to identify AT patients and/or AT heterozygotes accurately or to classify patients into various complementation groups. The instant invention provides a clear direction for AT research and the means to identify mutations in ATDC genes. Identifying such mutations provides for methods to diagnose AT, preferably AT-D, and to detect AT heterozygotes, preferably AT-D heterozygotes. Detection of AT heterozygotes is important because they have been reported to have an increased risk of cancer in response to treatment with ionizing radiation [Swift et al., N. Engl. J. Med., 325: 1831-1836 (1991)].